Uracil reductase inactivators

ABSTRACT

Uracil reductase inactivators, notably a 5-substituted uracil or 5,6-dihydro-5-substituted uracil, potentiate 5-flourouracil and find use particularly in the treatment of cancer. The 5-substituent is selected from bromo, ido, cyano, halo-substituted C- 1-4  alkyl, C 2-6  alkenyl, 1-halo-C 2-6  alkenyl and halo-substituted C 2-6  alkynyl.

[0001] The present invention relates to certain enzyme inactivatorswhich are useful in medicine, particularly cancer chemotherapy,especially in combination with antimetabolite antineoplastic agents suchas 5-fluorouracil (5-FU).

[0002] 5-Fluorouracil has been used in cancer chemotherapy since 1957.sensitive tumours include breast cancer, gastrointestinal malignancies,and cancers of the head and neck; 5-fluorouracil is also used as aradiation sensitiser. 5-Fluorouracil is metabolised rapidly in the liver(half life between about 8 and 20 minutes) by the enzymedihydropyrimidine dehydrogenase (uracil reductase). It has been reported(Cancer Research 46, 1094, 1986) that 5-(2-bromovinyl)-uracil (BVU) isan inhibitor of dihydrothymidine dehydrogenase which both retards themetabolism of 5-fluorouracil and enhances its antitumour activity. Ithas been reported that 5-(2-bromovinyl)-2′-deoxyuridine (which ismetabolized in vivo to BVU) enhances the antitumour activity of5-fluorouracil and 5-deoxy-5-fluorouridine, a prodrug of 5-fluorouracil(Biochemical Pharmacology 38; 2885, (1989)).

[0003] Unfortunately BVU is toxic to humans.

[0004] It has now been discovered that a group of 5-substituted uracilderivatives are inactivators of uracil reductase; they increase thelevel and half life of 5-fluorouracil in plasma and enhance the activityof 5-fluorouracil. They also reduce the normally encountered variationsof 5-fluorouracil plasma levels between subjects.

[0005] Accordingly, in a first aspect, the present invention provides auracil reductase inactivator which is a 5-substituted- or5,6-dihydro-5-substituted-uracil derivative, wherein the 5-substituentis bromo, iodo, cyano, halo-substituted C-₁₄ alkyl, C₂₋ ₆ alkenyl, a1-halo C₂₋₆ alkenyl group, a C₂₋₆ alkynyl group, a halo-substituted C₂-₆alkynyl group, or a prodrug thereof, for use in medicine, particularlyfor use in cancer chemotherapy. The uracil reductase inhibitor willgenerally be used in conjunction with 5-fluorouracil or a prodrugthereof.

[0006] By a C₂-₆ alkynyl group is meant a straight or branched chainalkynyl group, the latter including an alkynyl group substituted by acycloalkyl group containing between 2 and 6 carbon atoms in total.

[0007] The halogen substituent on the alkenyl or alkynyl group ispreferably bromo, chloro or iodo. Halo-substituted ethenyl and ethynylgroups are particularly preferred. Usually only one halo substituentwill be present.

[0008] In a further aspect, the present invention provides a uracilderivative as hereinbefore defined for use in the manufacture of amedicament for use in cancer chemotherapy. The medicament may also beuseful for rescue from 5-fluorouracil toxicity; and together with5-fluorouracil or a prodrug thereof for the treatment of psoriasis orrheumatoid arthritis, or human papilloma virus infections.

[0009] In a further aspect, the present invention provides a method forthe treatment or prophylaxis of tumours which comprises theadministration of an effective amount of uracil derivative ashereinbefore defined in the treatment of tumours in mammals, includingman. Preferably the treatment is in combination with 5-fluorouracil or aprodrug thereof.

[0010] In a yet further aspect, the present invention provides acombination of a uracil derivative as hereinbefore defined or prodrugthereof, and 5-fluorouracil or a prodrug thereof.

[0011] Preferred uracil derivatives are these wherein the 5-substituentis a C₂₋₆ alkynyl group (optionally halo-substituted), conveniently aC₂₋₄ alkynyl group and preferably an ethynyl or propynyl group. Inpreferred 1-halo-alkenyl and alkynyl derivatives the multiple bond is inthe 1-position. Particularly preferred inactivators of uracil reductasefor use in accordance with the invention are 5-ethynyluracil and5-propynyluracil. Other inactivators for such use include:

[0012] 5-cyanouracil

[0013] 5-bromoethynyluracil

[0014] 5-(1-chlorovinyl)uracil

[0015] 5-iodouracil

[0016] 5-hex-1-ynyluracil

[0017] 5-vinyluracil

[0018] 5-trifluoromethyluracil

[0019] 5-bromouracil

[0020] Uracil derivatives where the 5-substituent is a substituted orunsubstituted C₃-₆ alkynyl group are novel compounds and form a furtheraspect of the present invention.

[0021] Prodrugs of the uracil derivatives hereinbefore defined arecompounds which may be metabolised in vivo to give the uracilderivatives. These prodrugs may or may not have activity in their ownright but will normally have little activity. Such prodrugs includenucleoside analogues which contain a nucleobase corresponding to theabove 5-substituted uracil compounds, for example nucleoside derivativescontaining a ribose, 2′-deoxyribose, 2′, 3′-dideoxyribose, arabinose orother cleavable sugar portion, which may additionally contain a 2′ or3′-substituent such as halo, e.g. chloro or fluoro; alkoxy; amino orthio. Specific examples of such nucleoside derivatives are1-(b-D-arabinofuranosyl)-5-prop-1-ynyluracil; and2′,3′-dideoxy-5-ethynyl-3′-flourouridine. Compounds analogous toprodrugs of 5-FU as mentioned hereafter may in general be employed.References herein to uracil derivatives (or uracil reductaseinactivators) include reference to prodrugs thereof.

[0022] Prodrugs of 5-fluorouracil (5-FU) are compounds which aremetabolised in vivo to 5-fluorouracil and include 5-fluorouridine,5-fluoro-2-deoxyuridine, 5-fluoro-2-deoxycytidine,5′-deoxy-4′,5-fluorouridine, 5¹-deoxy-5-fluorouridine,1-(2-tetrahydrofuranyl)-5-fluorouracil and 1-C₁₋₈alkylcarbamoyl-5-fluorouracil derivatives.

[0023] 5-FU or a prodrug thereof and the said 5-uracil derivative may beemployed in combination in accordance with the invention byadministration of the components of the combination to an appropriatesubject either concomitantly, for example in a unitary pharmaceuticalformulation; or, more preferably, separately or sequentially within asufficient time period whereby the desired therapeutic effect of thecombination is achieved. Preferably the 5-uracil derivative isadministered first, and 5-FU or a prodrug thereof administeredsubsequently, advantageously from 15 mins to four days, usually 1 to 15hours, especially 1 to 2 hours thereafter.

[0024] 5-FU or a prodrug thereof and the 5-uracil derivative may beadministered respectively for therapy by any suitable route includingoral, rectal, nasal, topical (including buccal and sublingual), vaginaland parenteral (including subcutaneous, intramuscular, intravenous andintradermal). It will be appreciated that the preferred route will varywith the condition and age of the recipient, the nature of the infectionand other clinical factors.

[0025] Hitherto it has not been viable to administer 5-FU orally, as itis destroyed by uracil reductase in the gastro-intestinal tract.However, it has now been found that if a 5- substituted uracilderivative (as hereinbefore defined) is administered prior to oraladministration of 5-FU (or a prodrug thereof), high and persistentlevels of 5-FU are obtained in the plasma, indicating that this compoundis not being destroyed. This is a further advantage of the presentinvention. Preferably the 5-FU is administered within 15 mins to fourdays, usually 1 to 15 hours, especially 1 to 2 hours of the 5-uracilderivative.

[0026] Normally, patients exhibit a high degree of variability in 5-FUplasma concentrations resulting from a given 5-FU dosage, which may bedue to rates of 5-FU elimination which differ from patient to patient.There may also be diurnal variations within individual patients. The useof the 5-substituted uracil derivative according to the presentinvention is found to markedly reduce this subject-to-subjectvariability (see Experiment 3).

[0027] In general a suitable dose of 5-FU or a prodrug thereof will bein the range of 0.1 to 1000 mg per kilogram body weight of the recipientper day, preferably in the range of 0.1 to 200 mg per kilogram bodyweight per day. If 5-FU itself is administered the dose is preferably inthe range of 0.1 to 50 mg per kilogram body weight per day but higherdoses of prodrugs of 5-FU may be administered. The dose of 5-FU orprodrug thereof may be administered in unit dosage forms, for example,containing 5 to 3000 mg, preferably 20 to 1000 mg, active ingredient perunit dosage form.

[0028] Experiments with 5-FU suggest that a dose should be administeredto achieve peak plasma concentrations of the active compound of fromabout 0.01 to about 1.5 ug/ml.

[0029] The 5-uracil derivative may be administered in a dosage in therange of 0.01 to 50 mg per kilogram body weight of the recipient perday, particularly 0.01 to 10 mg/kg. The dose is more preferably in therange of 0.01 to 0.4 mg per kilogram body weight per day, depending onthe derivative used. An alternative preferred administration regime is0.5 to 10 mg/kg once per week.

[0030] The desired dose is preferably presented as one, two or moresub-doses administered at appropriate intervals throughout the day.These sub-doses may be administered in unit dosage forms for examplecontaining 1 to 200 mg preferably 2 to 100 mg, more preferably 2 to 50mg, of the 5-uracil derivative.

[0031] The uracil reductase inactivator and the 5-FU are usuallyemployed in an appropriate ratio to substantially reduce the naturalsubsisting uracil reductase level in the subject. Such a ratio based onthe respective weights of uracil reductase inactivator and 5-FU isgenerally in the range 1:0.01 to 1:100, preferably in the range 1:0.1 to1:50, and particularly in the range 1:1 to 1:10.

[0032]5-FU or prodrug thereof and the 5-uracil derivative are preferablyadministered in a pharmaceutical formulation, either in a singlepharmaceutical formulation containing both components or in separateadministrations each containing one of the components of thecombinations. The 5-uracil derivative will potentiate 5-FU, so thatlower doses of 5-FU will be employed.

[0033] The present invention thus includes as a further feature apharmaceutical formulation comprising a 5-uracil derivative ashereinbefore defined optionally in combination with 5-FU or a prodrugthereof together with at least one pharmaceutically acceptable carrieror excipient.

[0034] Each carrier must be “pharmaceutically acceptable” in the senseof being compatible with the other ingredients of the formulation andnot injurious to the patient. Formulations include those adapted fororal, rectal, nasal, topical (including buccal and sublingual), vaginaland parenteral (including subcutaneous, intramuscular, intravenous andintradermal) administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. Such methods include the step of bringinginto association the active ingredient with the carrier whichconstitutes one or more accessory ingredients. In general, theformulations are prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers or finely dividedsolid carriers or both, and then if necessary shaping the product.

[0035] Formulations of the present invention adapted for oraladministration may be presented as discrete units such as capsules,cachets or tablets each containing a predetermined amount of the activeingredient; as a powder or granules; as a solution or a suspension in anaqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion ora water-in-oil liquid emulsion. The active ingredient may also bepresented as a bolus, electuary or paste. Oral administration is thepreferred route.

[0036] A tablet may be made by compression or molding, optionally withone or more accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder (e.g. povidone, gelatin, hydroxypropylmethylcellulose),lubricant, inert diluent, preservative, disintegrant (eg. sodium starchglycollate, cross-linked povidone, cross-linked sodiumcaroxymethylcellulose) surface-active or dispersing agent. Moldedtablets may be made by molding in a suitable machine a mixture of thepowdered compound moistened with an inert liquid diluent. The tabletsmay optionally be coated or scored and may be formulated so as toprovide controlled release of the active ingredient therein using, forexample, hydroxypropylmethylcellulose in varying proportions to providethe desired release profile.

[0037] Formulations for topical administration in the mouth includelozenges comprising the active ingredient in a flavoured basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the active ingredient in asuitable liquid carrier.

[0038] Formulations for rectal administration may be presented as asuppository with a suitable base comprising for example cocoa butter ora salicylate.

[0039] Formulation for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

[0040] Formulations for parenteral administration include aqueous andnon-aqueous isotonic sterile injection solutions which may containanti-oxidants,- buffers, bacteriostats and solutes which renderthe-formulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be presented inunit-dose or multi-dose sealed containers, for example, ampoules andvials, and may be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid carrier, for examplewater for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets of the kind previously described.

[0041] Liquid formulations including dissolved 5-uracil derivative arepreferably buffered to a pH of 7 to 11, generally 9.5 to 10.5. Preferredunit dosage formulations are those containing a daily dose or unit,daily sub-dose, as hereinabove recited, or an appropriate fractionthereof, of an active ingredient.

[0042] The above-mentioned 5-uracil derivatives which are employed incombination with 5-fluorouracil or a prodrug thereof in accordance withthe present invention may be prepared in conventional manner. Forexample, the inactivators referred to above may be prepared by themethods described in J. Heterocycl. Chem. 19(3) 463-4 (1982) for thepreparation of 5-ethynyluracil; J.Chem. Soc. Perkin Trans. 1(16),1665-70 (1981) for the preparation of 5-(2-bromovinyl)uracil,5-bromoethynyluracil and 5-(2-bromo-1-chlorovinyl)uracil; Nucleic AcidChemistry, Vol. 2, 927-30 (1978) for the preparation 5-cyano-uracil;Nucleic Acids Research, 1(1) 105-7 (1974) for the preparation of5-vinyluracil; Z. Chem 17(11) 415-16 (1977) for the preparation of5-trifluoromethyluracil; Nucleic Acids Research 3 (10), 2845 (1976) forthe preparation of 5-(1-chlorovinyl)uracil.

[0043] The above prodrug nucleoside derivatives may also be prepared inconventional manner, for example in accordance with processes describedin European Patent Specification No. 356166 for the preparation of3′-fluoro-2′, 3′-dideoxy-5-alkynyluridine compounds, such as2′,3′-dideoxy-5-ethynyl-3′-fluorouridine, and European PatentSpecification No.272065 for the preparation of 5-alkynyluracilarabinosides, such as 1-(b-D-arabinofuranosyl) -5-prop-1-ynyluracil.

[0044] The novel 5-C₃₋₆ alkynyluracil compounds referred to above, whichare preferred 5-uracil derivatives for use in accordance with theinvention, may be prepared by one of the following processes, namely:

[0045] a) treatment of 5-C3-6 alkynyluridine compound to effectconversion thereof to be desired uracil compound; or

[0046] b) treatment of uracil compound substituted in the 5-position byan appropriate leaving group with a C₃-₆ alkyne to give the desireduracil compound.

[0047] In the above process a), conversion may be effected by enzymaticmeans, for example by treatment of the uridine compound with a thymidinephosphorylase enzyme, advantageously in a buffered medium at a pH of 6to 8.

[0048] In the above process b), a uracil compound substituted in the5-position by a suitable leaving group e.g. iodo or bromo, is treatedwith a C₃₋₆ alkyne in the presence of an appropriate palladium catalystsuch as bis (triphenylphosphine) palladium (II) chloride and cuprousiodide in an amine solvent such as triethylamine.

[0049] The following Examples illustrate the present invention.

EXAMPLE 1 5-Propynyluracil

[0050] A) To a stirred solution of 2′-deoxy-5-propynyluridine (EuropeanPatent Specification No. 272065) (20g, 75 mmol) in aqueous phosphatebuffer at pH 6.84 (1250 mL) was added purified E.coli thymidinephosphorylase (10,000 units) (T. A. Krenitsky et al, Biochemistry, 20,3615, 1981; U.S. Patent Specification No. 4,381,344) and alkalinephosphatase (10,000 units) [Sigma type VII-S from bovine intestinalmucosa] and the whole mixture was incubated at 37° C. for 24 hours. Theresulting white precipitate was filtered, washed with water (3×100 mL),ethanol (2×100 mL), ether (2×100 mL) and dried in vacuo over phosphoruspentoxide to give the title compound.

[0051] M.pt.: 275-280° C. (dec.)

[0052]¹H nmr & (d₆DMSO) 11.5-11.0 (bs, 2H, NH), 7.61 (1H, s, H-6), 1.95ppm (3H, s, CH₃)

[0053] Microanalysis calculated for C₇H₆N₂O₂: C,56.00;

[0054] H,4.03; N,18.66 Found: C,55.92; H,4.05; H,18.77

[0055] B) 1-Arabinofuranosyl-5-propynyluracil, (2.92 g, 20.4 mmoles),200 ml aqueous potassium phosphate, pH 6,8 4,000 IU thymidinephosphorylase (Krenitsky, T. A. et al Biochemistry, 20,3615,1981 and USPatent 4,381,444), 4,000 IU uridine phosphorylase (Krenitsky, T. A. etal Biochemistry, 20,3615,1981 and US Patent 4,381,444) and 2,000 IUalkaline phosphatase (Boehringer Mannheim) were stirred at 40° C. forfive days. Then 8,000 IU of thymidine phosphorylase, 20,000 IU uridinephosphorylase, 2,000 IU alkaline phosphatase and 30 IU acid phosphatase(Boehringer Mannheim) were added and incubationcontinued for anadditional five days. 5-Propynyluracil, being less soluble than thenucleoside, precipitated from the reaction mixture.

[0056] The precipitate and liquid were dried in vacuo, then5-propynyluracil was crystallized twice from hot water and vacuum driedat room temperature to give 0.92 g (6.1 mmoles) 5-propynyluracil in 59%yield.

[0057]¹H NMR & (dDMSO) 11.2 ppm (bs, 2H, 1H and 3H), 7.6 ppm (1H, s,6H), 1.95 ppm (3H, s CH ₃).

[0058] CHN calculated for C₇H₆N₂O₂: C, 56.00; H, 4.03; N, 18.66

[0059] Analyzed at: C, 55.95; H, 4.03; N, 18.60.

[0060] UV spectra: in 0.1 M HCl max at 287 nm and 231 nm; in 50 mMpotassium phosphate, pH 7.0 max at 287 nm and 231 nm; in 0.1 M NaOR maxat 306nm and 240 nm.

[0061] Mass spectrum gave peak at molecular ion weight of 151.

EXAMPLE 2 (5-ethynyluracil (EU))

[0062] (a) 5-(Trimethylsilylethynyl)uracil

[0063] A solution of 5-iodouracil (8g, 30mmol) in redistilledtriethylamine (500 mL) and dry DMF (10 mL) was degassed with oxygen-freenitrogen for 15 minutes. Bis(triphenylophosphine)palladium (II) chloride(0.5g), copper (I) iodide (0.5 g) and trimethylsilylacetylene (10 g, 102mmol) were then added and the mixture was heated with stirring at 50° C.for 24 hours. The cooled reaction mixture was filtered, the filtrateevaporated to dryness and the residue dissolved in dichloromethane (500mL). The organic solution was washed with a 2% aqueous solution ofdisodium EDTA (3×250 mL), water (3×200 mL), dried (Na₂SO₄) andevaporated to dryness. The residue was triturated with ethanol to givethe first crop of the title compound. The solid filtered from thereaction mixture was also found to contain the required product but in amore impure form and so was worked up as above in a separate batch togive a second crop.

[0064]¹H nmr & (d₆DMSO) 11.75-10.85 (2H, bs, NH), 7.75 (1H, 5, H-6),0.15 ppm (9H, m, SiCH₃).

[0065] (b) 5-Ethynyluracil

[0066] A solution of 5-(trimethylsilylethynyl)uracil (5.3 g, 24.4 mmol)in 0.2M solution of sodium methoxide in methanol (400 mL) was stirred atroom temperature for 3 hours and neutralized to pH 7 with dilutehydrochloric acid. The precipitated product was filtered, washed withmethanol and dried to give a first crop of the title compound. Thefiltrates and washings were combined, evaporated to dryness and theresidue crystallised from methanol to give the second crop of product.Combination of both crops and a further recrystallisation from ethanolgave a pure product.

[0067] M.pt.: 260° C. (dec.)

[0068]¹H nmr & (d₆DMSO) 11.6-10.8 (2H, bs, NH), 7.8 (1H, s, H-6), 4.03ppm (1H, s, acetylenic H)

[0069] Microanalysis calculated for C₆H₄N₂O₂ : C, 52.95; H, 2.96; N,20.58

[0070] Found: C, 52.04; H, 2.92; N, 20.3

EXAMPLE 3 (5-ethynyluracil)

[0071] a) 2,4-Dimethoxy-5-iodo-pyrimidine

[0072] A dry IL round-bottomed flask was charged with 5-iodouracil (50g, 0.21 mol), phosphorus oxychloride (300 ml), and N,N-diethylaniline (6drops). The heterogenous mixture was heated in a 120° C. oil bath undera nitrogen atmosphere for 24 hours. The phosphorus oxychloride wasdistilled off (some product co-distills off). The reaction solution wasnext slowly and cautiously poured over ice (1 L) and solid sodiumbicarbonate keeping the internal temperature at or below −20° C. (Thiswas accomplished by cooling in a dry-ice acetone bath). Once theaddition was complete, the reaction mixture was adjusted to pH 7 byaddition of solid sodium bicarbonate. The mixture was extracted withmethylene chloride and the organic fractions dried by passage throughphase separator paper. The crude solution of2,4-dichloro-5-iodopyrimidine was immediately added dropwise to asolution containing MeOH (400 ml) and sodium methoxide (28.8 g, 0.533mol). This addition took 1 hour. The reaction was then stirred at roomtemperature overnight. The solution was neutralized with CO₂(gas),extracted with methylene chloride, dried over anhydrous Na₂SO₄, filteredand concentrated. The crude product was adsorbed onto silica gel (100 g)and loaded onto a 400 g silica gel flash chromatography column. Thecolumn was eluted with 90:10 hexanes: ethyl acetate (v:v). Theappropriate fractions were combined and concentrated to a white solid asthe title compound.

[0073] Yield 26.7 g (48%)

[0074] 200MHZ NMR CDCl₃ &=3.97 (s, 3H); 4.02 (s, 3H), 8.43 (s, 1H).

[0075] b) 2,4-Dimethoxy-5-(b-trimethylsilyl)-ethynylpyrimidine

[0076] A dry 1L round-bottomed flask under a nitrogen atmosphere wascharged with the product of stage a) (26.7 g, 0.10 mol), dry methylenechloride (Aldrich, 150 mL), dry Et₃N (freshly distilled from KOHpellets, 250 mL). The system was evacuated and purged with nitrogenseveral times via a Firestone valve. Trimethylsilylacetylene (21.2 mL,0.15 mol; Aldrich) was added by syringe. Next were addedbis(triphenylphosphine)palladium (II) chloride (Aldrich 5.84 g, 8.32mmol) and copper (1) iodide (Aldrich 4.76 g, 25 mmol). The mixture washeated in a 60° C. oil bath for 2 hours, cooled and filtered throughCelite. The filtrate was concentrated in vacuo. The residue was dilutedwith toluene (100 mL) and then the toluene was removed in vacuo. Theresidue was taken up into methylene chloride (200 mL), filtered and thefiltrate extracted with 5% aq. ethylenediaminetetraacetic acid, disodiumsalt dihydrate (3×100 mL Aldrich), H₂O (1×100 mL). The organic layer wasdried via passage through phase separator paper and concentrated invacuo. The product was purified on a Waters Prep 500 eluting with 95:5hexanes: ethyl acetate (v:v). The crude product was adsorbed onto 100 gof silica gel and loaded onto a 400 g silica gel flash chromatographycolumn. The column was eluted with 97.5:2.5 hexanes: ethyl acetate(v:v). The appropriate fractions were combined and concentrated.

[0077] Yield 16.94 g (73%).

[0078] A 1.2 g sample of the resulting compound was bound to 6 g ofsilica gel and loaded onto a 50 g flash chromatography column. Thecolumn was eluted with hexanes: ethyl acetate 95:5 (v:v). Theappropriate fractions were combined, concentrated, stripped with CH₂Cl₂(2×30 mL), and dried in vacuo to yield 1.000 g of the title compound,m.p. 72.5-73° C.

[0079] Lit. m.p. 73-74° C. J. Heterocyclic Chem., 19, 463 ( 1982

[0080] c) 5-(b-trimethylsilyl)ethynyluracil

[0081] A dry 3-necked round-bottomed flask under nitrogen was chargedwith 2,4-dimethoxy-5-(b-trimethylsilyl)ethynylpyrimidine (6.5 g, 27.5mmol), dry acetonitrile (120 mL Aldrich), sodium iodide (oven dried invacua 80° C., 18 h, 12.4 g, 82.7 mmol) and chlorotrimethylsilane (10.5mL, 82.7 mmol freshly distilled). The mixture was heated at reflux for 3hours and then concentrated in vacua. The residue was digested with asolution containing methanol (40 mL) and water (20 mL) and the productfiltered off to give 1.48 g (26%). The product was dissolved inchloroform and the solution adsorbed onto silica gel 7 g) which was thenloaded onto a 35 g silica gel flash chromatography column. Elution withchloroform:methanol 95:5 (v:v) followed by chloroform:methanol 90:10(v:v) and evaporation of the product-containing fractions yielded 1.23 gof the title compound as a white solid.

[0082] d) 5-Ethynyluracil

[0083] A solution containing 5-(b-trimethylsilyl)ethynyluraci (3.85 g,18.4 mmol) and methanol (370 mL) was treated with a second solutioncontaining sodium hydroxide (2.3 g, 57.5 mmol) and water (1 mL). Themixture was stirred at room temperature for 2 hours and thenconcentrated in vacua The residue was suspended in water (35 mL) and thepH adjusted to 5 using 0.1 N HCl. The solids dissolved and then a secondprecipitate formed when the pH=5. The product was filtered, washed withH₂O, and then dried in vacuo to give 2.3 g (92%) of 5-ethynyluracil as alight beige powder.

[0084] Microanalysis calculated for C₆H₄N₂O₂: C, 52.95: H, 2.96; N,20.58

[0085] Found: C, 52.79; H, 3.02; N, 20.44

EXAMPLE 4 (5-ethynyluridine)

[0086] a) 2′,3′-5′-Tri-O-Acetyl-5-iodouridine

[0087] A dry 250 mL round-bottomed flask was charged with 5-iodouridine(10 g, 27 mmol Aldrich), anhydrous pyridine (30 mL) and acetic anhydride(30mL). The reaction was stirred at room temperature for 30 minutesunder a nitrogen atmosphere and the solvent removed in vacuo. Thecompound was diluted with toluene (2×50 mL) and the toluene removed invacuo. The product was purified on a 75 g flash chromatography columnwhich was eluted with 90:10 (v:v) CHCl₃:MeOH. The appropriate fractionswere combined and concentrated to give the title compound as a whitefoam. This was used directly in the next stage.

[0088] b) 2′,3′,5′-Tri-O-Acetyl-5-[2-(trimethylsilyl)ethynyl]uridine

[0089] A dry 1L round-bottomed flask equipped with a reflux condenser(under N₂ atmosphere) was charged with the product of stage a) (27mmol), dry methylene chloride (260 mL, Aldrich) and dry triethylamine(260 mL. freshly distilled from NaOH pellets). The system was evacuatedand purged with nitrogen several times and remained under a nitrogenatmosphere. Next was added (trimethylsilyl)acetylene (11.65 mL, 82 mmol;Aldrich) followed by copper (I) iodide (Aldrich, 1.57 g, 8.2 mmol) andbis(triphenylphosphine)palladium II chloride (Aldrich, 1.85 g, 2.6mmol). The mixture was heated in a 60° C. oil bath for 30 minutes,cooled, and filtered. The filtrate was concentrated in vacuo. Theresidue was taken up into CH₂Cl₂ (300 mL), filtered, washed with 5% aq.ethylenediaminetetraacetic acid, disodium salt (2×75 mL), H₂O (100 mL),dried over Na₂SO₄, filtered and concentrated in vacuo.

[0090] The resulting compound was bound to 50 g of silica gel and loadedonto a 400 g silica gel flash chromatography column which was elutedwith CHCl₃. The product fractions were combined and concentrated toyield the title compound as light yellow foam.

[0091] Yield 13 g

[0092] 300 MHz NMR CDCl₃ & 8.2 (br s, NH, 1H), 7.77 (s, 1H, H6), 6.11(d, H1′, 1H), 2.22 (s, 3H, OAc), 2.13 (s, 3H OAc), 2.11 (s, 3H, OAc),0.22 (s, 9H, SiMe₃).

[0093] c) 5-Ethynyluridine

[0094] The product of stage b) (9.5 g, 24 mmol) was dissolved inmethanol (200 mL) and diluted with a solution containing sodium (0.8 g)and methanol (100 mL). The reaction was stirred at room temperature for2 hours and was then neutralized using Dowex 50W-X8 (H⁺ form) resin. Theresin was removed by filtering and washed with methanol. The filtratewas concentrated in vacuo to give 4.85 g of a beige solid. The compoundwas purified on a Waters Prep 500 reverse phase C₁₈ column which waseluted with H₂O/MeOH 85:15 (v:v) to give 1.2 g of the title product(white solid). Impure fractions were re-chromatographed. An additional1.94 g of product were obtained.

[0095] Yield 49%

[0096] Calculated: % C,49.25 % H,4.47 % N,10.44

[0097] Found: % C,49,07 % H,4.53 % N,10.32

[0098] 200 MHz NMR (DMSOd₆) & 11.60 (br s, NH, 1H), 8.36 (s, H6, 1H),5.72 (d, J=4.3 Hz H1′, 1H), 4.01 (s, 1H, C═C—H).

[0099] The following Examples illustrate pharmaceutical formulations inwhich the “Active Ingredient” is 5-propynyluracil, 5-ethynyluracil orother uracil reductase inactivator as mentioned above; or mixturesthereof with 5-fluorouracil.

EXAMPLE 5 Tablet Formulations

[0100] The following formulations 5A, 5B and 5C are prepared by wetgranulation of the ingredients (except the magnesium stearate) with asolution of the povidone followed by drying of the granules, addition ofthe magnesium stearate and compression. Formulation 5A mg/tabletmg/tablet Active ingredient 5 2 Lactose, B.P. 205 75 Povidone, B.P. 1510 Sodium starch glycollate 20 10 Magnesium stearate 5 3 250 100

[0101] Formulation 5B mg/tablet mg/tablet Active ingredient 5 2 Lactose,B.P. 155 — Avicel PH 101 50 25 Povidone, B.P. 15 10 Sodium starchglycollate 20 10 Magnesium stearate 5 3 250 50

[0102] Formulation 5C mg/tablet Active ingredient 5 Lactose, B.P. 205Starch 50 Povidone, B.P. 6 Magnesium stearate 4 270

[0103] The following formulation 5D was prepared by direct compressionof the admixed ingredients. The lactose used is of the directcompression type. Formulation 5D mg/tablet Active ingredient  5 Lactose155 Avicel PH 101 100 260

[0104] The following formulation 5E is a controlled release tablet andis prepared by wet granulation of the ingredients (except magnesiumstearate) with a solution of the povidone, followed by drying of thegranules, addition of the magnesium stearate and compression.

EXAMPLE 6 Capsule Formulations

[0105] The following formulations 6A and 6B are prepared by admixing theuncompressed ingredients and filling into a two-part hard gelatincapsule. Formulation 6A mg/capsule Active ingredient 10 Lactose, B.P.250 Sodium starch glycollate 25 Magnesium stearate 5 290

[0106] Formulation 6B mg/capsule Active ingredient  5 Pregelatinizedstarch NF15 245 250

[0107] Formulation 6C mg/capsule Active ingredient  10 Macrogol 4000,B.P. 340 350

[0108] The Macrogol 4000, B.P. is melted and the active ingredientdispersed therein. The thoroughly mixed melt is then filled into atwo-part hard gelatin capsule.

EXAMPLE 7

[0109] Injectable Formulation Active ingredient 10 mg Sterile, pyrogenfree 10 ml pyrophosphate buffer (pH 10), q.s. to

[0110] The active ingredient is dissolved in most of the phosphatebuffer (35-40° C.), then made up to volume and filtered through asterile micropore filter into a 10 ml amber glass vial (type 1) andsealed with a sterile closure and overseal.

EXAMPLE 8

[0111] Suppository Formulation mg/suppository Active ingredient, 63 um* 10 Hard fat, B.P. (Witepsol H15-Dynamit Noble 1) 1790 1800

[0112] Our-fifth of the Witepsol H15 is melted in a steam-jacketed panat 45° C. maximum. The active ingredient is sifted through a 200 umsieve and added to the molten base with mixing, using a silverson fittedwith a cutting head, until a smooth dispersion is achieved. Maintainingthe mixture at 45° C., the remaining Witepsol H15 is added to thesuspension and stirred to ensure a homogeneous mix. The entiresuspension is passed through a 250 um stainless steel screen and, withcontinuous stirring, is allowed to cool to about 40° C. At a temperatureof 38° C. to 40° C. 1.80 g of the mixture is filled into suitableplastic molds. The suppositories are allowed to cool to roomtemperature.

[0113] Certain Experiments were carried out into the effectiveness of5-substituted uracils according to the invention.

Experiment 1

[0114] Determination of Uracil Reductase Inactivation Uracil reductase(1 micromolar) (dihydropyrimidine dehydrogenase, EC 1.3.1.2) purifiedfrom bovine liver was incubated with 100 micromolar inactivator and 5mMdithiothreitol (enzyme reductant) at 370 for 30 minutes in 0.05 MTris-HCl at pH 8.0. The enzyme and inactivator were diluted 100-foldinto the assay buffer, which contained 200 micromolar NADPH, 200micromolar thymine and 1 mM dithiothreitol in Tris-HCl at pH 8.0. Thevelocity of the enzyme was determined spectrophotometrically. Thesevelocities have been corrected for HADPH oxidase activity, which wasless than 10% of the rate of thymine-dependent oxidation of NADPH. The %inactivation of the enzyme was equal to 100% minus the percent ofenzymatic activity remaining. Enzyme incubated without inhibitor wasstable under these conditions. Parenthetical values are the relativefirst-order rate constants for inactivation of enzyme determined fromsimilar experiments where the fractional activity was measured as afunction of the time of incubation of 50 micromol inactivator withenzyme. The results are given below: Compound % Inactivation5-ethynyluracil 100 (100) 5-cyanouracil^(a) 100  (14) 5-propynyluracil100  (8) 5-bromoethynyluracil 100  (8) 5-(1-chlorovinyl)uracil 100  (5)5-iodouracil 100  (4) 5-hex-1-ynyluracil^(a) 90 5-vinyluracil^(a,b) 865-trifluoromethyluracil 75 5-bromouracil 75

[0115] a The inhibition was reversible since enzyme treated with thisderivative slowly regained activity after a 100-fold dilution into theassay mixture.

[0116] b These nucleobases were generated in situ by treating therespective nucleosides with 40 units/ml of thymidine phosphorylase in 35mM potassium phosphate for 20 minutes prior to addition to uracilreductase. The parent nucleosides were not inactivators.

[0117] The effectiveness of 5-ethynyluracil (EU) was investigated and isreported in the following Experiments 2 to 4 and Figures wherein

[0118]FIG. 1 shows increased levels of uracil and thymine at a time offour hours following various oral EU doses in rats; and

[0119]FIG. 2 shows that EU increased plasma levels of 5-fluorouracil(5-FU). Mice were dosed either orally (p.o.) or interperitoneally (i.p.)with 5-FU. 5-ethynyluracil (EU) at 2 mg/kg was dosed i.p. 90 minutesprior to the 5-FU.

Experiment 2 Inactivation of Uracil Reductase (in vivo)

[0120] Mice, rats, dogs and monkeys dosed with small amounts of5-ethynyluracil (EU) rapidly developed greatly elevated plasma uraciland thymine levels. The maximum effect occurred at about 0.1 mg/kg p.o.in rats, at 0.5 to 1 mg/kg s.c. in mice, and at approximately 1 mg/kgintravenously (i.v.) in dogs and probably represents total inactivationof uracil reductase. These doses elevated mouse, dog, and rat plasmauracil from about 3 uM to about 50-60 uM. Plasma uracil decreased tonormal over 24 hr (half-life=10 hr). FIG. 1 shows the increased plasmalevels of uracil and thymine in the rat at a time of 4 hours followingvarious oral doses of 5-ethynyluracil, due to inactivation of uracilreductase. The ED₅₀ equals 0.01 mg/kg.

Experiment 3 Effect on Plasma-FU Level

[0121] Mice and rats pretreated with 5-ethynyluracil (EU) and then dosedwith FU sustained higher plasma levels of FU than mice not pretreated(FIG. 2). In addition, the usual variability in plasma FU in rats orallydosed with FU at 50 mg/kg was eliminated by EU pretreatment. The AUC ofthe plasma FU concentration-time curves were 41, 126 and 68 (ave=78±55%)versus 417, 446 and 426 (ave =430±3%) for nonpretreated and EUpretreated rats, respectively.

Experiment 4 Potentiation of the Antitumor Activity of 5-fluorouracil(5-FU) in Mice by 5-Ethynyluracil (EU)

[0122] Colon 38 tumor was implanted in mice on day zero. Mice (8 pergroup) were treated with 5-FU on days one through nine with the dosesindicated in Table 2 EU was dosed i.p. at 2 mg/kg 30 minutes prior to5-FU dosing where indicated. TABLE 2 % Mice Tumor Free on Day 17 Dose5-FU (mg/kg) FU i.p. plus EU FU i.p. FU p.o. plus EU FU p.o. 0.25 0 0.525 0 1 12.5 25 2 37.5 37.5^(a) 3 100 4 100 10 0 0 15 12.5 12.5 20 12.512.5 25 — 12.5 30 87.5^(a) 12.5

1. A pharmaceutical composition, comprising (i) an amount effective toenhance 5-fluorouracil activity of a 5-substituted- or5,6-dihydro-5-substituted- uracil derivative, wherein the substituent atthe 5 position is halo substituted C₁₋₄ alkyl, C₂₋₆ alkenyl, 1-halo-C₂₋₆alkenyl, or C₂₋₆ alkynyl, or a prodrug thereof.
 2. The pharmaceuticalcomposition of claim 1, wherein the uracil derivative is5-ethynyluracil.
 3. The pharmaceutical composition of claim 1, whereinthe uracil derivative is 5-propynyl uracil.
 4. The pharmaceuticalcomposition of claim 1, wherein the uracil derivative is a prodrugselected from nucleoside analogs of ribose, 2′-deoxyribose,2′-3′-dideoxyribose, or arabinose each optionally substituted with halo,alkoxy, amino, or thio.
 5. The pharmaceutical composition of claim 4,wherein the uracil derivative is 1-(β-D-arabinofuranosyl)-5-prop-1-ynyluracil or 2′-3′-dideoxy-5-ethynyl-3′-fluorouridine.